Hemagglutination Inhibition Assay

Overview

Hemagglutination Inhibition Assay is a technique used for the classification or sub-typing of hemagglutinating viruses. HI assay is quantitive so it's frequently applied to evaluate the antigenic relationships between different virus isolates of the same subtype. HAI assays are an inexpensive procedure that utilizes standard laboratory equipment and is less technical than other molecular detection methods being easily completed in a couple of hours.

Required materials

• 96-well microtiter plates

• Red blood cells

• Virus or bacterial antigen

• Serum samples

• PBS

• Sterile distilled water

• 1% BSA

• 0.05% tween 20

• Micropipettes

• V-bottom microtiter plate

• Shaker or rocking platform

Protocol

1. Add 25 μl of sterile distilled water to each well.

2. In the first wells add 25 μl virus or bacterial antigen.

3. Then add 25 μl of serum samples to the first row and serially dilute.

4. Add 25 μl of the 50% RBCs to watch the wells on the plate.

5. Leave the plate at room temperature for 30 minutes for its incubation period.

6. Mix the contents after the incubation period via gentle rocking.

Results

For results observe the wells for any hemagglutination in either the presence or absence of the RBCs. The HI titer is the reciprocal of the highest serum dilutions that inhibit RBCs' hemagglutination. Calculate the titer of each serum and record the results.